LOCUS       sequence-464113-        6515 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Heterologous protein expression of replicase polyprotein 1a (pp1a) 
            (ORF1a polyprotein) in Escherichia coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6515)
  TITLE     AI Proteins, Inc. plasmids
REFERENCE   2  (bases 1 to 6515)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pAIP4_2H2Z
FEATURES             Location/Qualifiers
     source          1..6515
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     73..90
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    105..244
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(170..189)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     330..352
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(346..537)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1346..2428)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     2390..2409
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2429..2506)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2712..2731
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2819..2838
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2819..2837
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2838..2862
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             2877..2899
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2922..2951
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             4194..4211
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4264..4282)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4278..4325
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4362..4817
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4449..4468)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4659..4680
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4909..5724)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(4909..5724)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     5632..5651
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5716..5735
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5846..6434
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6335..6354
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
       61 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt
      121 ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct
      181 ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc
      241 tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
      301 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
      361 gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga
      421 ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa
      481 tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga
      541 tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag
      601 gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg
      661 taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca
      721 gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca
      781 gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg
      841 gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct
      901 tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc
      961 tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg ccatgccggc
     1021 gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc
     1081 gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg
     1141 aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat
     1201 gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga
     1261 gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag
     1321 ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     1381 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca
     1441 gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc
     1501 cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt
     1561 tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta
     1621 ccgagatgtc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg
     1681 ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca
     1741 tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa
     1801 tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac
     1861 ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc
     1921 ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga
     1981 catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt
     2041 catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg
     2101 ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca
     2161 gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac
     2221 tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt
     2281 tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa
     2341 cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg
     2401 cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc
     2461 gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc
     2521 tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc
     2581 accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg
     2641 gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga
     2701 tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg
     2761 atgccggcca cgatgcgtcc ggcgtagagg atcgagatcg atctcgatcc cgcgaaatta
     2821 atacgactca ctatagggga attgtgagcg gataacaatt cccctctaga aataattttg
     2881 tttaacttta agaaggagat atacatatga tggcgaagat tcatcaccat catcaccatc
     2941 accatcacca cggtagcctg caagattcgg aagtcaacca agaagcaaag ccggaagtca
     3001 agccggaagt gaaaccggaa acccatatta acctgaaagt tagtgacggc agctctgaaa
     3061 ttttctttaa gatcaaaaag accacgccgc tgcgtcgcct gatggaagcg tttgccaaac
     3121 gtcagggcaa ggaaatggat agcctgcgtt tcctgtatga cggtattcgc atccaggcag
     3181 atcaagcgcc ggaagacctg gacatggaag acaacgacat cattgaagcc caccgtgaac
     3241 agattggcgg ctctggaagt ggatttagga aaatggcttt cccctcaggt aaggtcgagg
     3301 gctgcatggt gcaagttacc tgtggtacta cgactttaaa cggcctgtgg ctggatgata
     3361 ccgtatactg cccgcgtcac gtgatttgca ccgcggaaga catgctgaac ccgaattacg
     3421 aggacttgct tatccgtaaa agcaatcact ccttcctggt tcaagcggga aacgtgcaac
     3481 tccgcgtgat cggtcattct atgcagaact gtctgctgag actgaaggta gacacctcta
     3541 acccgaagac cccgaagtat aaattcgtgc gtattcagcc gggtcagacc ttctccgtgc
     3601 tggcgtgtta taacggctct ccgagcggcg tttaccagtg cgcaatgcgt cctaatcaca
     3661 ccatcaaagg ttcgttcctg aacggcagct gcggttccgt tggttttaat attgattatg
     3721 attgtgttag cttctgctac atgcaccaca tggaactgcc gaccggcgtt catgcaggca
     3781 ccgatttgga aggtaagttc tatggtccgt ttgtggaccg ccaaacggcc caagcggctg
     3841 gtacggatac gaccatcacc ctgaacgttt tggcgtggtt atatgcggcg gttatcaacg
     3901 gcgaccgttg gtttctgaac cgttttacca ccacgttgaa cgacttcaat ctggtggcta
     3961 tgaaatacaa ttacgagccg cttacccagg atcatgttga catcttgggc ccgctgagcg
     4021 cacagaccgg cattgctgtc ctggacatgt gcgccgctct gaaagaactg ttgcaaaacg
     4081 gtatgaatgg tcgtaccatc ctcggcagca ccattctgga ggacgagttc acgccatttg
     4141 atgtcgtgcg ccagtgcagc ggtgtgacct ttcagggttc gtaatgactc gagcaccacc
     4201 accaccacca ctgagatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg
     4261 ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt
     4321 ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct gtagcggcgc
     4381 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     4441 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     4501 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     4561 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     4621 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     4681 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     4741 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     4801 attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     4861 ttatttttct aaatacattc aaatatgtat ccgctcatga attaattctt agaaaaactc
     4921 atcgagcatc aaatgaaact gcaatttatt catatcagga ttatcaatac catatttttg
     4981 aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaag
     5041 atcctggtat cggtctgcga ttccgactcg tccaacatca atacaaccta ttaatttccc
     5101 ctcgtcaaaa ataaggttat caagtgagaa atcaccatga gtgacgactg aatccggtga
     5161 gaatggcaaa agtttatgca tttctttcca gacttgttca acaggccagc cattacgctc
     5221 gtcatcaaaa tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagcgag
     5281 acgaaatacg cgatcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg
     5341 caggaacact gccagcgcat caacaatatt ttcacctgaa tcaggatatt cttctaatac
     5401 ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac catgcatcat caggagtacg
     5461 gataaaatgc ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat
     5521 ctcatctgta acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc
     5581 atcgggcttc ccatacaatc gatagattgt cgcacctgat tgcccgacat tatcgcgagc
     5641 ccatttatac ccatataaat cagcatccat gttggaattt aatcgcggcc tagagcaaga
     5701 cgtttcccgt tgaatatggc tcataacacc ccttgtatta ctgtttatgt aagcagacag
     5761 ttttattgtt catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     5821 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     5881 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     5941 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag
     6001 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     6061 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     6121 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     6181 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     6241 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     6301 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     6361 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     6421 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     6481 cttttgctca catgttcttt cctgcgttat cccct
//
